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Background: Cardiac fibroblasts have crucial roles in the heart. In particular, fibroblasts differentiate into myofibroblasts in the damaged myocardium, contributing to scar formation and interstitial fibrosis. Fibrosis is associated with heart dysfunction and failure. Myofibroblasts therefore represent attractive therapeutic targets. However, the lack of myofibroblast-specific markers has precluded the development of targeted therapies. In this context, most of the noncoding genome is transcribed into long noncoding RNAs (lncRNAs). A number of lncRNAs have pivotal functions in the cardiovascular system. lncRNAs are globally more cell-specific than protein-coding genes, supporting their importance as key determinants of cell identity., Methods: In this study, we evaluated the value of the lncRNA transcriptome in very deep single-cell RNA sequencing. We profiled the lncRNA transcriptome in cardiac nonmyocyte cells after infarction and probed heterogeneity in the fibroblast and myofibroblast populations. In addition, we searched for subpopulation-specific markers that can constitute novel targets in therapy for heart disease., Results: We demonstrated that cardiac cell identity can be defined by the sole expression of lncRNAs in single-cell experiments. In this analysis, we identified lncRNAs enriched in relevant myofibroblast subpopulations. Selecting 1 candidate we named FIXER (fibrogenic LOX -locus enhancer RNA), we showed that its silencing limits fibrosis and improves heart function after infarction. Mechanitically, FIXER interacts with CBX4, an E3 SUMO protein ligase and transcription factor, guiding CBX4 to the promoter of the transcription factor RUNX1 to control its expression and, consequently, the expression of a fibrogenic gene program.. FIXER is conserved in humans, supporting its translational value., Conclusions: Our results demonstrated that lncRNA expression is sufficient to identify the various cell types composing the mammalian heart. Focusing on cardiac fibroblasts and their derivatives, we identified lncRNAs uniquely expressed in myofibroblasts. In particular, the lncRNA FIXER represents a novel therapeutic target for cardiac fibrosis., Competing Interests: Disclosures Dr Pedrazzini is cofounder of HAYA Therapeutics, Epalinges, Switzerland.

Evolutionary conservation is a measure of gene functionality that is widely used to prioritise long noncoding RNAs (lncRNA) in cancer research. Intriguingly, while updating our Cancer LncRNA Census (CLC), we observed an inverse relationship between year of discovery and evolutionary conservation. This observation is specific to cancer over other diseases, implying a sampling bias in the selection of lncRNA candidates and casting doubt on the value of evolutionary metrics for the prioritisation of cancer-related lncRNAs.

Long noncoding RNAs (lncRNAs) are widely dysregulated in cancer, yet their functional roles in cancer hallmarks remain unclear. We employ pooled CRISPR deletion to perturb 831 lncRNAs detected in KRAS-mutant non-small cell lung cancer (NSCLC) and measure their contribution to proliferation, chemoresistance, and migration across two cell backgrounds. Integrative analysis of these data outperforms conventional "dropout" screens in identifying cancer genes while prioritizing disease-relevant lncRNAs with pleiotropic and background-independent roles. Altogether, 80 high-confidence oncogenic lncRNAs are active in NSCLC, which tend to be amplified and overexpressed in tumors. A follow-up antisense oligonucleotide (ASO) screen shortlisted two candidates, Cancer Hallmarks in Lung LncRNA 1 ( CHiLL1 ) and GCAWKR , whose knockdown consistently suppressed cancer hallmarks in two- and three-dimension tumor models. Molecular phenotyping reveals that CHiLL1 and GCAWKR control cellular-level phenotypes via distinct transcriptional networks. This work reveals a multi-dimensional functional lncRNA landscape underlying NSCLC that contains potential therapeutic vulnerabilities., Competing Interests: T.P., R.E., and R.J. have filed a patent application (21202363.4) relating to the therapeutic targeting of lncRNAs identified here. Applicant: University of Bern. R.J. is a paid consultant for NextRNA (USA)., (© 2022 The Author(s).)

Human and other genomes encode tens of thousands of long noncoding RNAs (lncRNAs), the vast majority of which remain uncharacterised. High-throughput functional screening methods, notably those based on pooled CRISPR-Cas perturbations, promise to unlock the biological significance and biomedical potential of lncRNAs. Such screens are based on libraries of single guide RNAs (sgRNAs) whose design is critical for success. Few off-the-shelf libraries are presently available, and lncRNAs tend to have cell-type-specific expression profiles, meaning that library design remains in the hands of researchers. Here we introduce the topic of pooled CRISPR screens for lncRNAs and guide readers through the three key steps of library design: accurate annotation of transcript structures, curation of optimal candidate sets, and design of sgRNAs. This review is a starting point and reference for researchers seeking to design custom CRISPR screening libraries for lncRNAs., (© 2021. The Author(s).)

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays., (© 2022. The Author(s).)

Background: Obstructive sleep apnea (OSA) is prevalent in pregnancy and it is associated with adverse pregnancy-related outcomes such as gestational diabetes, pre-eclampsia, and low birth weight. Maternal systemic inflammation is proposed to be one of the main intermediate mechanisms. However, the effects of OSA on systemic inflammation are unknown in normal pregnancy. Methods: Women in the 3rd trimester underwent hospital polysomnography to evaluate whether OSA increases systemic inflammation in normal pregnancy and its potential association with adverse fetal outcomes. OSA was defined as an apnea-hypopnea index (AHI) of ≥ 5 h -1 . Plasma cytokines levels (TNF-α, IL-1β, IL-6, IL-8, and IL-10) were determined by multiple immunoassays. Results: We included 11 patients with OSA and 22 women with AHI < 5 h -1 , who were homogeneous in age, and body mass index (BMI). Women with OSA had significant higher levels of TNF-α, IL-1β, IL-8, and IL-10. We found significant correlations between AHI during REM and TNF-α ( r = 0.40), IL-1β ( r = 0.36), IL-6 ( r = 0.52), IL-8 ( r = 0.43), between obstructive apnea index and TNF-α ( r = 0.46) and between AHI and IL-1β ( r = 0.43). We also found that CT90% was related to IL-8 ( r = 0.37). There were no significant differences in neonatal characteristics; however, we found inverse correlations between TNF-α and IL-8 with birth weight (both r = -0.48), while IL-8 showed a significant inverse relationship with neonatal gestational age ( r = -0.48). Conclusions: OSA in our normal pregnancy population was associated with higher systemic inflammation, which was related to obstructive events, especially during REM sleep. Moreover, systemic inflammation was inversely correlated with neonatal birth weight and age., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alonso-Fernández, Ribot Quetglas, Herranz Mochales, Álvarez Ruiz De Larrinaga, Sánchez Barón, Rodríguez Rodríguez, Gil Gómez, Pía Martínez, Cubero Marín, Barceló Nicolau, Cerdà Moncadas, Codina Marcet, De La Peña Bravo, Barceló Bennasar, Iglesias Coma, Morell-Garcia, Peña Zarza, Giménez Carrero, Durán Cantolla, Marín Trigo, Piñas Cebrian, Soriano and García-Río.)

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-an early step in NHEJ-yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion., (© 2021 Bosch-Guiteras et al.; Published by Cold Spring Harbor Laboratory Press.)

Regulatory non-protein coding RNAs perform a remarkable variety of complex biological functions. Previously, we demonstrated a role of the human non-coding vault RNA1-1 (vtRNA1-1) in inhibiting intrinsic and extrinsic apoptosis in several cancer cell lines. Yet on the molecular level, the function of the vtRNA1-1 is still not fully clear. Here, we created HeLa knock-out cell lines revealing that prolonged starvation triggers elevated levels of apoptosis in the absence of vtRNA1-1 but not in vtRNA1-3 knock-out cells. Next-generation deep sequencing of the mRNome identified the PI3K/Akt pathway and the ERK1/2 MAPK cascade, two prominent signaling axes, to be misregulated in the absence of vtRNA1-1 during starvation-mediated cell death conditions. Expression of vtRNA1-1 mutants identified a short stretch of 24 nucleotides of the vtRNA1-1 central domain as being essential for successful maintenance of apoptosis resistance. This study describes a cell signaling-dependent contribution of the human vtRNA1-1 to starvation-induced programmed cell death.

Motivation: CRISPR-Cas9 loss-of-function (LOF) pooled screening promises to identify which long non-coding RNAs (lncRNAs), amongst the many thousands to have been annotated so far, are capable of mediating cellular functions. The two principal LOF perturbations, CRISPR-inhibition and CRISPR-deletion, employ one and two guide RNAs, respectively. However, no software solution has the versatility to identify hits across both modalities, and the optimal design parameters for such screens remain poorly understood., Results: Here, we present CRISPR Analysis for Single and Paired RNA-guides (CASPR), a user-friendly, end-to-end screen analysis tool. CASPR is compatible with both CRISPRi and CRISPR-del screens, and balances sensitivity and specificity by generating consensus predictions from multiple algorithms. Benchmarking on ground-truth sets of cancer-associated lncRNAs demonstrates CASPR's improved sensitivity with respect to existing methods. Applying CASPR to published screens, we identify two parameters that predict lncRNA hits: expression and annotation quality of the transcription start site. Thus, CASPR is a versatile and complete solution for lncRNA CRISPR screen analysis, and reveals principles for including lncRNAs in screening libraries., Availability and Implementation: https://judithbergada.github.io/CASPR/., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Long non-coding RNAs (lncRNAs) represent a huge reservoir of potential cancer targets. Such "onco-lncRNAs" have resisted traditional RNAi methods, but CRISPR-Cas9 genome editing now promises functional screens at high throughput and low cost. The unique biology of lncRNAs demands screening strategies distinct from protein-coding genes. The first such screens have identified hundreds of onco-lncRNAs promoting cell proliferation and drug resistance. Ongoing developments will further improve screen performance and translational relevance. This Review aims to highlight the potential of CRISPR screening technology for discovering new onco-lncRNAs, and to guide molecular oncologists wishing to apply it to their cancer of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We combine the labeling of newly transcribed RNAs with 5-ethynyluridine with the characterization of bound proteins. This approach, named capture of the newly transcribed RNA interactome using click chemistry (RICK), systematically captures proteins bound to a wide range of RNAs, including nascent RNAs and traditionally neglected nonpolyadenylated RNAs. RICK has identified mitotic regulators amongst other novel RNA-binding proteins with preferential affinity for nonpolyadenylated RNAs, revealed a link between metabolic enzymes/factors and nascent RNAs, and expanded the known RNA-bound proteome of mouse embryonic stem cells. RICK will facilitate an in-depth interrogation of the total RNA-bound proteome in different cells and systems.

Background: In patients with breast cancer who are candidates for neoadjuvant therapy (NAT), the timing of when to perform sentinel lymph node biopsy (SLNB) remains under discussion. The aim of this study was to compare the advantages and disadvantages of SLNB performed before and after NAT., Patients and Methods: One hundred seventy-two patients, T1c to T3 and N0 (clinically and according to ultrasound) candidates for NAT were included. We compared the outcomes of 2 groups: (1) 122 patients of whom SLNB was performed before NAT (pre-NAT) from December 2006 to April 2014; and (2) 50 patients with SLNB performed after NAT (post-NAT) from May 2014 to July 2016., Results: Both groups were homogeneous in baseline patient characteristics. The SLNB was positive in 50 patients [41.7%] (33 macrometastases [66%] and 17 micrometastases [34%]) versus 6 patients [12%] (5 macrometastases [83.3%] and 1 micrometastases [16.7%]) in pre- and post-NAT groups, respectively. The lymphadenectomy was performed in 34 patients [28.3%] versus 4 patients [8%], with an odds ratio of 3.48 (95% confidence interval, 1.3-9.3). The recurrences in the pre-NAT group after a median follow-up of 62 months were 12 systemic, 2 local and systemic, and none axillary. In the post-NAT group were no recurrences after a median follow-up of 16 months. Finally, SLNB after NAT reduces the delay in starting NAT from 24 to 14 days (medians; P < .001) and the identification of the SLNB was in 122 patients [100%] versus 49 patients [98%]., Conclusion: SLNB performed after NAT significantly reduces the rate of lymphadenectomies without any increase in recurrences at early follow-up. Furthermore, it allows systemic treatment to be started earlier without interfering in the SLNB identification rate., (Copyright © 2017 Elsevier Inc. All rights reserved.)

CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.

Background and Objective: It remains controversial whether sentinel lymph node biopsy (SLNB) should be performed before or after neoadjuvant therapy (NAT). We aimed to evaluate the feasibility and accuracy of SLNB before NAT at a single institution, and to determine its relation to patient prognosis., Methods: A prospective study of T1c-T2-T3 N0 breast cancer patients, after ultrasound examination, who underwent SLNB prior to NAT. Overall, disease-specific and disease-free survival were calculated by Kaplan-Meier curves., Results: SLNB before NAT was performed in 123 patients from December 2006 to May 2014. The identification rate was 100%. SLNB was positive in 42.3% of cases (27.6% macrometastases). NAT was chemotherapy in 88.6% of cases and endocrine-therapy in 11.4%. Lymphadenectomy was avoided in 72.4% of cases. Median follow-up was 40 months (range 8-100). Overall and disease-free survival was 90.2% and 88.6% respectively.SLN involvement was not related to patient outcome (p 0.72); however there were significant differences in survival according to molecular-like subtypes (p < 0.025) and NAT response (p < 0.0001)., Conclusions: SLNB prior to NAT is an accurate method of axillary staging associated with a high identification rate. It avoided lymphadenectomy in more than 70% of patients. SLN involvement did not worsen the prognosis in our cohort., (Copyright © 2017 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.)

Background: The objective of this study was to determine stress levels during hospitalization in patients with Chronic Obstructive Pulmonary Disease (COPD). We wanted to relate stress to previous level of quality of life and patients' Social Support., Methods: 80 patients (70.43; SD = 8.13 years old) with COPD were assessed by means of: Hospital Stress Rating Scale, Nottingham Health Profile, St. George's Respiratory Questionnaire and Social Support Scale., Results: COPD patients' stress levels are lower than expected independently from the severity or number of previous hospitalizations. Linear regression analysis shows the predictive value of Quality of Life and Social Support on stress level during hospitalization (p < 0.0001)., Conclusion: HRQOL and social support can be associated with stress during hospitalization.

Unlabelled: Chronic Obstructive Pulmonary Disease (COPD) is a progressive disease with dyspnoea perception as a main symptom. In severe stages, dyspnoea can constitute a risk factor for depression, anxiety and somatization disorders., Objective: The objective was to evaluate the presence of these psychopathologies based on dyspnoea and severity stages in patients with COPD., Materials and Methods: Patients (n = 51) were evaluated by means of the Hospital Anxiety and Depression Scale, the dyspnoea scale (MRC), the General Health Questionnaire (GHQ-28) and spirometric criteria., Results: The increase in dyspnoea level and disease severity lead to a progressive worsening of anxiety, depressive and somatic symptoms with clinical relevance (P < 0.05). There was a significant correlation between those parameters (P < 0.05)., Conclusions: The early detection and treatment of these psychopathologies associated with dyspnoea and progression of the disease must be taken into account in this complex pathology., (Copyright © 2010 SEGG. Published by Elsevier Espana. All rights reserved.)